Minimization or Withdrawal of Immunosuppression in Liver Transplantation

Transcription

1 Trends in Transplantation 2007;1:15-23Alberto Sánchez-Fueyo: Operational Tolerance in Liver Transplantation Minimization or Withdrawal of Immunosuppression in Liver Transplantation Alberto Sánchez-Fueyo Liver Unit, Hospital Clinic, University of Barcelona, IDIBAPS, Barcelona, Spain Abstract Human liver allografts have a lower susceptibility to than other organs. In addition, in some liver transplant recipients immunosuppressive drugs can be completely withdrawn and these patients are considered as operationally tolerant. Accumulated clinical experience indicates that elective immunosuppressive drug weaning is feasible in almost 20% of selected liver transplant recipients. This is associated with an incidence of 12-76% of acute cellular, but these episodes are commonly mild and often resolve by return to baseline immunosuppression, many times without the need to administer steroid boluses. The study of tolerance in liver transplantation has been hampered by the absence of prospective studies correlating the results of immune-monitoring assays and clinical outcome. Thus, we lack a clinically validated treatment-stopping rule capable of predicting the success of immunosuppression and this procedure has to be performed on a trial and error basis. The search for an accurate means to identify allograft tolerance among immunosuppressed recipients should become a priority in liver-transplantation research. This information would provide a biologic basis for guiding immunosuppression- protocols and for the implementation of tolerance-promoting strategies in liver transplantation. (Trends in Transplant 2007;1:15-23) Corresponding author: Alberto Sánchez-Fueyo, AFUEYO@clinic.ub.es Key words Liver transplantation. Tolerance. Immunosuppression. Operational tolerance. Regulatory lymphocites. Correspondence to: Alberto Sánchez-Fueyo Liver Unit Hospital Clinic, University of Barcelona, IDIBAPS Villarroel, Barcelona, Spain AFUEYO@clinic.ub.es 15

2 Trends in Transplantation 2007;1 Introduction Liver allografts are unique in that indefinite survival in the absence of immunosuppressive therapy can be achieved in pigs, rats, and mice 1-3. In these animals, acute occurs but resolves spontaneously, and recipients can accept organs from the same donor but not from third-party donors 3. The unique immuno-privileged status of liver allografts is also evident in clinical transplantation, and has been exemplified in many clinical conditions such as: resistance to positive cross-match; irrelevance of human leukocyte antigen (HLA) matching; reduced incidence of hyperacute ; immunomodulating effect of the liver in case of combined hepatorenal grafting; occasional spontaneous recovery following a severe episode; irrelevance of a single episode in relation to later graft outcome; reduced incidence of chronic and reversal of established chronic in 30% of treated patients; achievement of very similar clinical results regardless of whether aggressive or simple immunosuppression schemes are employed; the ease with which the stage of steroidfree immunosuppression can be reached 4-6. Moreover, successful of all immunosuppression is possible in selected patients, as has been shown accidentally in noncompliant patients, and purposely sought in patients presenting with posttransplant lymphoproliferative disease (PTLD), life-threatening infections, or in patients enrolled in carefully monitored prospective immunosuppression-weaning protocols. These patients off all immunosuppressive drugs have been shown to remain healthy in some cases for more than 30 years 7 and are therefore considered as operationally tolerant. Altogether it is clear from the aforementioned clinical data that livers exhibit unique tolerogenic properties, and are therefore the allografts most amenable to immunosuppression minimization or and probably to tolerance induction. The underlying mechanisms of this intrinsic tolerogenic ability are not fully understood and multiple factors have been proposed. The liver produces large amounts of soluble major histocompatibility complex (MHC) class I antigens 8, and these soluble antigens have been shown to induce apoptosis of alloreactive CD8+ T-cells both in vitro and in vivo, leading to prolonged transplant survival 9,10. The exchange of migratory leukocytes between the graft and the recipient leading to long-term cellular microchimerism has also been proposed as a basis for the acceptance of liver allografts 11. However, although donor-cell microchimerism can occasionally be detected in humans long after organ transplantation 12, most studies have failed to establish a link between donor-cell persistence and tolerance maintenance A conceptually different hypothesis involving donor-derived passenger leukocytes postulates that tolerogenic dendritic cells (DC), generated from bone marrow-derived precursors contained within the graft, are central in the acceptance of liver allografts by suppressing cytopathic immune responses and promoting regulatory mechanisms Similar effects could be mediated by other liver-derived antigen-presenting cells, including liver sinusoidal endothelial cells 19 and liver macrophages (Kupffer cells). Furthermore, the size of the liver, its un-barricaded anatomical structure, and its anti-inflammatory cytokine microenvironment may add to its tolerogenic properties by promoting immune-cell trafficking and ensuring optimal opportunities for engagement with parenchymal and non-parenchymal liver cells. One of the downstream consequences of these tolerance-promoting mechanisms appears to be the extensive elimination by apoptosis of graft-infiltrating alloreactive T-cells 21, a phenomenon widely documented in experimental models Other cellular elements required to ensure liver allograft acceptance in experimental models are naturally occurring CD4+ CD25+ regulatory T-cells. Despite this con- 16

3 Alberto Sánchez-Fueyo: Operational Tolerance in Liver Transplantation siderable amount of data gathered from experimental models, none of the abovementioned mechanisms have been unambiguously validated in humans yet. Definition of allograft tolerance The use of the term tolerance in the field of transplantation is somewhat confusing, and multiple definitions have been employed over time in the clinical literature. From an immunologic point of view, tolerance defines a state of immune non-reactivity towards a specific set of antigens that is indefinitely maintained in the absence of ongoing immunosuppression. In experimental models, tolerance is induced through therapies aiming at the deletion of alloreactive cytopathic T-cells and/or generation of regulatory T-cells, and is formally proved by the demonstration that tolerant recipients accept same-donor second grafts without further immunosuppression, while rapidly rejecting third-party grafts. In addition, in tolerant experimental animals, T-cells often display anti-donor hyporesponsiveness, and in cases of peripheral tolerance in which regulatory T-cells are involved, tolerance can be transferred to naive hosts by T-cell adoptive transfer. These formal demonstrations of tolerance are obviously unsuitable for clinical application. Hence the term operational tolerance was chosen to designate the clinical situation in which a graft maintains a stable function without features of acute or chronic and without the need for chronic immunosuppression. All intentional immunosuppression-weaning trials published so far have employed operational tolerance as their clinical endpoint. Although this is correct from a clinical perspective, it should not lead us to assume that operationally tolerant patients are immunologically equivalent to experimentally tolerant rodents. Indeed, we still have a very incomplete understanding of the mechanisms responsible for allograft tolerance in humans, and lack for instance a detailed picture of the nature of donor-specific immune responses in these patients. In fact, even the extent to which operationally tolerant patients constitute an immunologically homogeneous population is unknown. A totally different concept is the notion of minimal immunosuppression, which is commonly applied to identify patients who, after having been treated mostly but not exclusively with potent T-cell depleting antibodies at the time of transplantation, are capable of maintaining stable graft function under the cover of very low doses of calcineurin inhibitors 6,25,26. Minimal immunosuppression is also known as prope or near tolerance, although it is not clear at all whether minimally immunosuppressed patients actually resemble operationally tolerant recipients, and whether they will ever be capable of completely discontinuing immunosuppression without developing. Clinical experience in immunosuppression after liver transplantation The first observations of drug-free tolerance in liver transplantation (LT) were reported in 1993 by Starzl, who described the cases of six noncompliant patients having normal liver function five to 13 years after transplantation 12. Since this landmark report, an increasing clinical experience has been gathered, although publications regarding immunosuppression remain scarce. Critical evaluation of the actual reality of tolerance after LT requires that distinctions be made, first between temporary and definitive immunosuppression, and second between non-elective weaning due to noncompliance, which involves non-selected patients, and elective or planned weaning, a slow process addressed to well-selected patients. Clinical experience with temporary immunosuppression The published experience with immunosuppression discontinuation in the face of life-threatening infections or PTLD has been summarized in table 1. Collectively, the combined published ex- 17

4 Trends in Transplantation 2007;1 perience amounts to 108 patients, mostly children One of the lessons that emerge from these reports is that temporary immunosuppression is possible and in most cases is not immediately followed by acute. Furthermore, although it is not uncommon for to eventually occur (in around 35% of patients), it can be reversed by resumption or reinforcement of immunosuppression with a very small rate of graft loss. In fact, immunosuppression reinstitution is often not performed until is formally proven. Most likely, inhibition of cellular immunity by severe sepsis or PTLD acts as a facilitator of successful immunosuppression. In short, management of posttransplant life-threatening complications employing complete immunosuppression cessation may be warranted, although in most cases immunosuppression needs to be progressively reinitiated as the general condition of the recipient improves. In the group of patients suffering from Epstein-Barr virus (EBV)-related PTLD the proportion of successful immunosuppression weaning appears to be higher than in other circumstances. Whether less-aggressive immunosuppression reduction could achieve similar patient survival outcomes remains an open question. Clinical experience with elective immunosuppression During the period , results from intentional immunosuppression trials were reported by the Pittsburgh, King s College London, Kyoto, Murcia, New Orleans, Rome, and Miami groups 14, Data from these reports are summarized in table 2. Collectively, the accumulated experience amounts to 304 patients. Some of the particularities of each series are discussed below. Pittsburgh experience Immunosuppression was electively withdrawn in 95 (33% pediatric) selected recipients grafted between 1992 and At the time data Table 1. Temporary immunosuppression in liver transplantation Time between IS and Graft loss due to Acute/ chronic Maintenance IS at the time of IS Follow-up after IS Time between LT and IS (days) Cause of IS No. of patients Year Author Reference number 22/11% 0 TAC + steroids (11) CsA + steroids (7) 140 days (median) 17 days (median) 1994 Mañez R (31)* Lifethreatening infection or PTLD 25/0% 0 mean: 35 days range: days CsA + steroids + AZA 66 days (median) 32 days (mean) (adults) Lifethreatening infection 1998 Massarollo PC TAC + steroids 35.3/0% 0 mean: 240 days range: days PTLD in 30 NA 24 months (median) 2001 Takatsuki M (children) 50/2.6% 2.6% mean: 107 days range:7-476 days TAC + steroids in > 90% patients 51 months (mean) 272 days (median) PTLD in 19; EBV in Hurwitz M (children) *This report provides information from 31 patients, but only 18 of them survived more than 20 days. 15 patients (44%) remained off IS for a median of 24 months. 8 patients (21%) remained off IS for a mean of 4.2 years. 18

5 Alberto Sánchez-Fueyo: Operational Tolerance in Liver Transplantation Table 2. Complete elective immunosuppression in liver transplantation Year Author Reference number No. of patients Selection criteria* Maintenance IS Time between LT and weaning (years) Complete IS Follow-up after IS (months) Acute/ Chronic Graft loss due to 1997 Mazariegos JV (30% children) > 5 years post-lt > 2 years without (biopsy proven) 13.7 % AZA 11.6% TAC 75% CsA mean: 8.4 range: % median: 35.5 range: / 0 % Devlin R Girlanda R (adults) > 5 years post-lt Side effects of chronic IS CsA and AZA median: 7 range: % /5.6% 5.6% Takatsuki M Oike F (children) > 2 years post-lt Normal liver function tests > 1 year without TAC > % median: 21.9 range: /0% Pons JA 31 9 (adults) > 2 years post-lt CsA median: 5.2 range: % range: /0% Eason JD (adults) > 6 months post-lt Normal liver function tests TAC > % 12 61/0% Tryphonopoulos P (adults) > 3 years post-lt > 12 months without No autoimmune disease 85% TAC 14% CsA mean: 4 range: % mean: 25.8 range: %/1.9% 0.96% 2006 Tisone G (adults) > 1 year post-lt HCV-RNA positive Absence of or cirrhosis on basal biopsy CsA mean: % mean: 45.5 range: /0% 0 IS: immunosuppression; LT: liver transplantation; TAC: tacrolimus; CsA: cyclosporin A; AZA: azathioprine; HCV: hepatitis C virus. *In all cases absence of recurrent original liver disease (other than HCV infection) was required for enrolment. All patients were living donor liver transplant recipients. 19

6 Trends in Transplantation 2007;1 20 were submitted for publication, results were as follows: 18 (19%) patients were off immunosuppression; 37 (39%) patients were on spaced immunosuppression; 28 (29%) patients underwent acute ; and 12 patients were withdrawn from the protocol. There was a highly significant advantage for azathioprine (AZA) or tacrolimus (TAC) baseline immunosuppression at the time of weaning as compared to cyclosporin A (CsA). It is important to note that although 46% of patients had liver-test elevations, acute was present in only 25 of them (26%, 18 cases biopsy proven and seven diagnosed based on high clinical suspicion). The mean delay between the start of weaning and biopsy proven acute was 13.2 months, most episodes were mild, and in all cases they were easily reversed using steroid pulses (83%) or switch to TAC. No patient developed chronic, although in three patients minor duct injuries were detected prompting immunosuppression resumption. All 18 patients off immunosuppression had a significantly improved health-related quality of life, and in patients developing during the weaning procedure the average immunosuppression at the end of a three-year follow-up was less than at their entry in the study. A later report from the same group analyzing peripheral blood obtained from six pediatric liver recipients off immunosuppression showed that, compared to patients under maintenance immunosuppression, successfully weaned patients had increased numbers of potentially tolerogenic plasmacytoid dendritic cells (pdc), and decreased numbers of the theoretically more immunogenic myeloid dendritic cell (mdc) subset (increased pdc/mdc ratio) 36. This finding, however, remains to be validated in view of more recent reports showing that both increasing age and chronic immunosuppression diminish peripheral blood pdc numbers 37,38. King s College experience In 18 adults with an uneventful post-lt follow-up of more than five years, immunosuppression drugs were abruptly discontinued under strict in-hospital monitoring 14. After a follow-up of more than three years, five patients (27%) remained off immunosuppression, while in the remaining 13 elevated liver tests were detected and immunosuppression was eventually recommenced. Important again was the fact that out of these 13 patients with deranged liver tests only four had acute on liver biopsy, with only one case of severe among them. Acute was easily reversed using steroid pulses (77%) or switch to TAC. In eight patients, liver biopsies revealed a hepatitis-like disorder with a mixed inflammatory portal-tract infiltrate, occasionally also affecting the lobules. Predictors of successful immunosuppression were: lower incidence of early post-lt, good HLA matching, non-autoimmune and nonviral primary liver diseases, but not presence of donor microchimerism. In a recent addendum to their first report, out of the five patients successfully withdrawn from immunosuppression, one patient originally transplanted for primary sclerosing cholangitis developed chronic and required regrafting, while in another one, immunosuppression was restarted due to low-grade 39. Kyoto experience The Kyoto experience relates to a cohort of 26 pediatric, living, related LT recipients, having one HLA haplotype identity, in whom immunosuppression was electively weaned 33. Immunosuppression was successfully withdrawn in six patients (23%). At the end of follow-up 15.4% of patients had encountered acute (successfully resolved in all cases with reintroduction of TAC or steroid boluses) and 36.5% were still being weaned. In a later update 40 the number of patients included in elective weaning was increased to 67 with almost identical results (23.8% success and 12% acute ). No cases of hepatitis-like disorders were reported. Neither the degree of HLA matching nor early episodes of was found to predict success. In contrast, more recent reports from the same group have described that downregulation of intra-graft

7 Alberto Sánchez-Fueyo: Operational Tolerance in Liver Transplantation TH1 cytokines 41 and increased numbers of peripheral blood regulatory CD4+ CD25+ T-cells and delta-1 gamma-delta T-cells are associated with the immunosuppression-free tolerant state 42. The Kyoto group has very recently communicated that in two out of 11 (18%) operationally tolerant liver recipients in whom protocol liver biopsies were performed an average of four years after weaning, substantial bile-duct atrophy was detected; reintroduction of TAC showed histologic recovery 43. This important observation should be compared with a previous report from Sebagh, indicating that among patients on maintenance immunosuppression more than 10 years after transplantation and exhibiting normal liver function tests, histologic signs of chronic can be detected in 24% of cases 44. Altogether, these reports suggest that operationally tolerant liver recipients, similarly to patients requiring ongoing immunosuppression, might not be completely protected from the development of chronic. Murcia experience Nine elective patients were included in a prospective immunosuppression-weaning protocol. Three patients could be completely taken off immunosuppression, while significant elevations of liver-function tests occurred in the remaining patients due to acute (two patients) and a mixed inflammatory portal tract infiltrate (four patients) 31. Liver endothelial cell chimerism was studied in five patients and not found to be associated to successful weaning. New Orleans experience The recent report by Eason is unusual in that sustained immunosuppression was attained in only one (5.5%) out of 18 patients 32. It must be highlighted that in this study immunosuppression weaning was started earlier than in any other study; indeed weaning was offered after just six months post-lt. In addition, the reasons for the discontinuation of the immunosuppression-weaning procedure in some patients are not clearly reported. In this series there was one episode of steroid-resistant that required thymoglobulin administration. Miami experience The Miami group investigated the effect of donor bone-marrow infusions administered early postoperatively on the complete of immunosuppression at least three years after transplantation 19. Successful weaning was achieved in 19% (20 patients out of 104) regardless of whether recipients had received donor bone marrow (45 patients) or not (59 patients). Two patients developed chronic, and one of them required retransplantation for this reason. No significant differences were found in donor cell chimerism levels (performed in bone-marrow aspirates using PCR flow) between successfully weaned patients and those developing graft s. It should be noted that in most patients acute was diagnosed on a clinical basis without the performance of liver biopsy. Rome experience This series is also particular in that it relates to 34 hepatitis C virus (HCV) positive patients with recurrent allograft disease 34. Immunosuppression was successful in eight patients (23.5%). Among the remaining patients, 12 developed acute during the weaning procedure, while in 14 patients acute occurred during the eight months after immunosuppression (in one of them was detected 43 months after). No episodes of severe were detected and no treatment other than CsA resumption was required. Remarkably, successful immunosuppression was associated with stabilization or improvement of histologic fibrosis. Low CsA through levels during the first week of LT and steroid-free immunosuppression were both found to be predictors of successful weaning. 21

8 Trends in Transplantation 2007;1 22 Barcelona experience At Hospital Clínic Barcelona we have recently reported the results of an immune-profiling study performed on a cohort of operationally tolerant liver recipients gathered from University Tor Vergata, UCL-Brussels, Hospital Vall d Hebró, and our own Liver Transplant Unit. Our data confirm the results from the Kyoto group in terms of the increased numbers of peripheral blood CD4+ CD25+ T-cells and delta-1 gamma-delta T-cells. In addition, our report provides data on a gene expression pattern characteristic of the tolerant state. These results can be regarded as a first step in the search for a diagnostic test of operational tolerance in liver transplantation 45. Concluding remarks The available data indicate that elective immunosuppression is possible in 19.4% of recipients. Favorable clinical markers for successful immunosuppression appear to be at least two years of post-lt followup, low incidence of previous acute episodes, non-autoimmune primary liver disease, and possibly minimized post-lt immunosuppression. There are, however, two main caveats that have to be kept in mind when interpreting these published studies. The first one has to do with the selection criteria employed for enrolment in immunosuppression protocols. These criteria have differed depending on the series (Table 2), and in some but not all studies, patients have been selected for weaning precisely on the basis of likelihood of successful immunosuppression. For this reason, and in the absence of an intent-to-treat weaning trial, it is difficult to accurately estimate the actual rate of operational tolerance in LT. The second limitation is the absence in many series of follow-up reports providing long-term clinical and histologic data. This information is critical in order to ascertain the robustness and duration of the tolerant state. The incidence of documented acute cellular during immunosuppression weaning ranges from 12 to 76%, but these episodes are in most cases mild, and often resolve by return to baseline immunosuppression with or without administration of steroid boluses. Reassuringly, only two cases of graft loss (due to chronic ) among patients involved in immunosuppression-weaning protocols have been reported. Considering the absence of biochemical predictors of, liver biopsies are required at baseline and during the weaning procedure, and maybe even during the follow-up after successful immunosuppression. The significance of the high incidence (up to 78% in some series) of hepatitis-like disorders detected in liver biopsies obtained during weaning is not clear. These findings, consisting of portal and lobular necroinflammation, were already reported more than ten years ago by Pappo in long-term LT recipients on maintenance immunosuppression 46, and probably constitute a form of late, since many improve after immunosuppression resumption In conclusion, immunosuppression weaning can be considered in stable and wellselected long-term patients and in patients presenting with immunosuppression-related life-threatening complications. However, since no reliable biomarkers are yet able to identify tolerance, this approach still needs to be performed on a trial and error basis. Thus, the generation of a robust, clinically applicable, diagnostic algorithm of allograft tolerance is urgently needed. References 1. Kamada N, Calne RY. A surgical experience with 530 liver transplants in the rat. Surgery 1983;93: Kamada N. Animal models of hepatic allograft. Semin Liver Dis 1992;12: Qian S, Demetris AJ, Murase N, Rao AS, Fung JJ, Starzl TE. Murine liver allograft transplantation: tolerance and donor cell chimerism. Hepatology 1994;19: Markus BH, Duquesnoy RJ, Gordon RD, et al. Histocompatibility and liver transplant outcome. Does HLA exert a dualistic effect? Transplantation 1988;46: Calne R, Davies H. Organ graft tolerance: the liver effect. Lancet 1994;343: Lerut JP. Avoiding steroids in solid organ transplantation. Transpl Int 2003;16: Demetris AJ, Eghtesad B, Marcos A, et al. Recurrent hepatitis C in liver allografts: prospective assessment of diagnostic accuracy, identification of pitfalls, and observations about pathogenesis. Am J Surg Pathol 2004;28: Mathew JM, Shenoy S, Phelan D, Lowell J, Howard T, Mohanakumar T. Biochemical and immunological evaluation of donorspecific soluble HLA in the circulation of liver transplant recipients. Transplantation 1996;62: Scherer MN, Graeb C, Tange S, Dyson C, Jauch KW, Geissler EK. Immunologic considerations for therapeutic strategies utiliz-

9 Alberto Sánchez-Fueyo: Operational Tolerance in Liver Transplantation ing allogeneic hepatocytes: hepatocyte-expressed membranebound major histocompatibility complex class I antigen sensitizes while soluble antigen suppresses the immune response in rats. Hepatology 2000;32: Behrens D, Lange K, Fried A, et al. Donor-derived soluble MHC antigens plus low-dose cyclosporine induce transplantation unresponsiveness independent of the thymus by down-regulating T-cell-mediated alloresponses in a rat transplantation model. Transplantation 2001;72: Starzl TE, Demetris AJ, Murase N, Ildstad S, Ricordi C, Trucco M. Cell migration, chimerism, and graft acceptance. Lancet 1992; 339: Starzl TE, Demetris AJ, Trucco M, et al. Cell migration and chimerism after whole-organ transplantation: the basis of graft acceptance. Hepatology 1993;17: Wood KJ. Passenger leukocytes and microchimerism: what role in tolerance induction? Transplantation 2003;75:17-20S. 14. Devlin J, Doherty D, Thomson L, et al. Defining the outcome of immunosuppression after liver transplantation. Hepatology 1998;27: Buhler LH, Spitzer TR, Sykes M, et al. Induction of kidney allograft tolerance after transient lymphohematopoietic chimerism in patients with multiple myeloma and end-stage renal disease. Transplantation 2002;74: Lau AH, Thomson AW. Dendritic cells and immune regulation in the liver. Gut 2003;52: Lau AH, de Creus A, Lu L, Thomson AW. Liver tolerance mediated by antigen presenting cells: fact or fiction? Gut 2003;52: Gassel HJ, Hutchinson IV, Engemann R, Morris PJ. The role of T suppressor cells in the maintenance of spontaneously accepted orthotopic rat liver allografts. Transplantation 1992;54: Knolle PA, Schmitt E, Jin S, et al. Induction of cytokine production in naive CD4+ T-cells by antigen-presenting murine liver sinusoidal endothelial cells but failure to induce differentiation toward Th1 cells. Gastroenterology 1999;116: Khanna A, Morelli AE, Zhong C, Takayama T, Lu L, Thomson AW. Effects of liver-derived dendritic cell progenitors on Th1- and Th2-like cytokine responses in vitro and in vivo. J Immunol 2000;164: Qian S, Lu L, Fu F, et al. Apoptosis within spontaneously accepted mouse liver allografts: evidence for deletion of cytotoxic T-cells and implications for tolerance induction. J Immunol 1997;158: Crispe IN. Hepatic T-cells and liver tolerance. Nat Rev Immunol 2003;3: Crispe IN, Dao T, Klugewitz K, Mehal WZ, Metz DP. The liver as a site of T-cell apoptosis: graveyard, or killing field? Immunol Rev 2000;174: Bishop GA, Wang C, Sharland AF, McCaughan G. Spontaneous acceptance of liver transplants in rodents: evidence that liver leucocytes induce recipient T-cell death by neglect. Immunol Cell Biol 2002;80: Calne RY. Prope tolerance: the future of organ transplantation from the laboratory to the clinic. Transplantation 2004;77: Starzl TE, Murase N, Abu-Elmagd K, et al. Tolerogenic immunosuppression for organ transplantation. Lancet 2003;361: Manez R, Kusne S, Linden P, et al. Temporary of immunosuppression for life-threatening infections after liver transplantation. Transplantation 1994;57: Massarollo PC, Mies S, Abdala E, Leitao RM, Raia S. Immunosuppression for treatment of severe infections in liver transplantation. Transplant Proc 1998;30: Hurwitz M, Desai DM, Cox KL, Berquist WE, Esquivel CO, Millan MT. Complete immunosuppressive as a uniform approach to posttransplant lymphoproliferative disease in pediatric liver transplantation. Pediatr Transplant 2004;8: Mazariegos GV, Reyes J, Marino IR, et al. Weaning of immunosuppression in liver transplant recipients. Transplantation 1997;63: Pons JA, Yelamos J, Ramirez P, et al. Endothelial cell chimerism does not influence allograft tolerance in liver transplant patients after of immunosuppression. Transplantation 2003; 75: Eason JD, Cohen AJ, Nair S, Alcantera T, Loss GE. Tolerance: is it worth the risk? Transplantation 2005;79: Takatsuki M, Uemoto S, Inomata Y, et al. Weaning of immunosuppression in living donor liver transplant recipients. Transplantation 2001;72: Tisone G, Orlando G, Palmieri G, et al. Complete weaning off immunosuppression in HCV liver transplant recipients is feasible and favorably impacts on the progression of disease recurrence. Journal of Hepatology 2006;44: Tryphonopoulos P, Tzakis AG, Weppler D, et al. The role of donor bone marrow infusions in of immunosuppression in adult liver allotransplantation. Am J Transplant 2005;5: Mazariegos GV, Zahorchak AF, Reyes J, et al. Dendritic cell subset ratio in peripheral blood correlates with successful of immunosuppression in liver transplant patients. Am J Transplant 2003;3: Shodell M, Siegal FP. Circulating, interferon-producing plasmacytoid dendritic cells decline during human ageing. Scand J Immunol 2002;56: Hackstein H, Renner FC, Bohnert A, et al. Dendritic cell deficiency in the blood of kidney transplant patients on long-term immunosuppression: results of a prospective matched-cohort study. Am J Transplant 2005;5: Girlanda R, Rela M, Williams R, O Grady JG, Heaton ND. Longterm outcome of immunosuppression after liver transplantation. Transplant Proc 2005;37: Oike F, Yokoi A, Nishimura E, et al. Complete of immunosuppression in living donor liver transplantation. Transplant Proc 2002;34: Takatsuki M, Uemoto S, Inomata Y, et al. Analysis of alloreactivity and intragraft cytokine profiles in living donor liver transplant recipients with graft acceptance. Transpl Immunol 2001; 8: Li Y, Koshiba T, Yoshizawa A, et al. Analyses of peripheral blood mononuclear cells in operational tolerance after pediatric living donor liver transplantation. Am J Transplant 2004;4: Ueda M, Koshiba T, Li Y, Pirenne J, Haga H, Tanaka K. Requirement of protocol biopsy before and after complete cessation of immunosuppression following living-donor liver transplantation. Liver Transpl 2006;11 [abstract 122]. 44. Sebagh M, Rifai K, Feray C, et al. All liver recipients benefit from the protocol 10-year liver biopsies. Hepatology 2003; 37: Martinez-Llordella M, Puig-Pey I, Orlando G, et al. Multi-parameter of immune profiling of operational tolerance in liver transplantation. Am J Transplant 2007;7: Pappo O, Ramos H, Starzl TE, Fung JJ, Demetris AJ. Structural integrity and identification of causes of liver allograft dysfunction occurring more than 5 years after transplantation. Am J Surg Pathol 1995;19: